The obligate intracellular bacterium Chlamydia trachomatis is a widely disseminated pathogen that infects epithelial surfaces of the conjuctiva and urogenital tract. Chronic inflammation from repeated and persistent infections can lead to severe complications such as blinding trachoma, pelvic inflammatory disease and infertility. Despite the clinical and public health importance of chlamydial diseases, the bacterium's intractability to mutational analysis has significantly hindered a molecular understanding of its pathogenesis. In this grant application, we propose to develop a methodology using chemical mutagens and DNA deep sequencing technologies to perform genetic analysis in Chlamydia. In Aim 1, we will identify mutations responsible for the formation of a "small plaque" (Spq) phenotype by backcrossing mutants to reference strains and following the co-segregation of single nucleotide polymorphisms (SNP) with the Spq phenotype. Putative causative mutations will be then identified by sequencing the minimal region of DNA that co-segregates with the Spq phenotype. In Aim2, we will define the essential gene set required for Chlamydia replication in tissue culture cells. Chromosomal DNA from a large set of independently derived, heavily mutagenized Chlamydia isolates will be sequenced in pools. We will assess the relative contribution of individual genes to Chlamydia viability by monitoring their ability to tolerate loss-of-function mutations. The proposed approaches will significantly accelerate the translation of Chlamydia genomic sequences into functional information and establish a blueprint for genetic analysis in other "genetically-intractable" pathogens. PUBLIC HEALTH RELEVANCE: Chlamydial infections are a significant public health burden and a high risk for the development of reproductive diseases. Our understanding of their biology and how they cause disease is limited by the lack of tools to perform genetic analysis. Here we propose to develop methods to perform such an analysis based on new DNA sequencing technologies. Results from this research will identify factors important for Chlamydia survival in the host and provide new targets for therapeutic intervention and vaccine design.